Alzheimer?s disease (AD) is a progressive neurodegenerative disease likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significance importance as APOE4 carriers account for 65-80% of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Emerging data suggests that apoE4 is sensitive to proteolytic cleavage and thus contributes to the underlying molecular pathology associated with AD possibly through a loss of function. Using a site-directed antibody to cleaved apoE4 we have recently determined an amino-terminal cleavage fragment of apoE4 of 17 KDa can be formed following incubation of full-length apoE4 with matrix metalloproteinase-9 (MMP-9) that localizes to the nucleus of microglia of the human AD brain. The goal of this proposal is to expand those findings by determining the mechanisms by which a recombinantly-produced, His-tagged fragment of apoE4 (nApoE41-151) is taken up by microglia, traffics to the nucleus, and alters gene expression. We hypothesize that the trafficking of this fragment to the nucleus serves a pathophysiological function that regulates the expression of genes related to microglia activation and cell death. Experiments described in Aim 1a will rigorously test the hypothesis that nApoE41-151 is taken up by cells through a specific receptor-mediated pathway involving the low-density lipoprotein (LDL) receptor or the LDL receptor-related protein (LRP-1). In Aim 1b, parallel experiments will also assess whether trafficking of nApoE41-151 to the nucleus occurs aided by the use of our site-directed cleavage antibody that specifically detects this apoE4 fragment as well as an anti-His antibody. We propose experiments to elucidate the pathway by which nApoE41-151 traffics from the cytoplasm to the nucleus, presumably following receptor-mediated endocytosis. Experiments outlined in Aim 2 will determine what transcriptional effects if any this apoE4 fragment has once localized within the nucleus. In Aim 2a, we will first characterize the potential binding of nApoE41-151 to a possible enhancer region of nuclear DNA 5? to a novel, uncharacterized gene that was isolated following chromatin immunoprecipitation in preliminary studies. These experiments will analyze the potential binding kinetics, in vitro, utilizing several techniques that allow for the characterization of interactions of the DNA sequence and nApoE41-151. In addition, in Aim 2b, we will determine if this sequence serves a potential regulator of transcription using a luciferase reporter gene construct. Finally, in Aim 2c we will examine overall gene expression in BV2 microglial cells following treatment with nApoE41-151 following purification of RNA samples and transcriptome analysis. Because microglia produce apoE4, our hypothesis is this fragment is part of a feedback loop that regulates the expression of APOE4 gene or may lead to transcriptional regulation of other genes that contribute to cell death or microglia activation. Data from this proposal could uncover a novel pathophysiological role for apoE4 and lead to a better understanding as to why inheritance of this gene enhances AD risk.